As a result of innovations in genetic engineering, medicines employing bio-related substances such as intercellular messengers, e.g., hormones and cytokines, antibodies, and enzymes have been developed and investigated actively in recent years. After having been administered to a living body, these bio-related substances usually disappear from the living body due to glomerular filtration within the kidneys and the uptake of macrophages within the liver, spleen, etc. The bio-related substances hence have a short half-life period in the blood, and it is difficult to obtain a sufficient pharmacological effect. In order to overcome this problem, attempts to improve the behavior in living body have been made by encapsulating a bio-related substance in liposomes or polymer micelles or increasing the molecular weight or forming a hydration layer through chemical modification with an amphipathic polymer such as a sugar chain or polyethylene glycol, or albumin. Modification with a polyoxyalkylene has brought about effects such as a reduction in toxicity or antigenicity and an improvement in the solubility of sparingly water-soluble drugs.
In recent years, techniques have been actively investigated in which when a bio-related substance is modified with a polyoxyalkylene, a target site on the bio-related substance is selectively modified with a desired number of polyoxyalkylene segments without deactivating the active sites of the bio-related substance. Furthermore, there is a trend toward increases in the molecular weights of polyoxyalkylene derivatives in order to obtain a larger hydration layer with a smaller number of polyoxyalkylene chains. In particular, there is a desire for a high-molecular polyoxyalkylene derivative having an oxyamino group which selectively reacts with an aldehyde or ketone group to form a relatively stable oxime bond.
JP-A-7-196925 includes a statement concerning reagents for forming PEG hydrazone and PEG oxime bonds and protein derivatives of these. International Publication No. 96/041813 includes a statement concerning a polyoxyalkylene derivative having an oxyamino group for use in site-selective reactions. Moreover, JP-T-2007-501811 (the term “JP-T” as used herein means a published Japanese translation of a PCT patent application) includes a statement concerning a technique in which trans-glutaminase is introduced into a peptide and this glutaminase is used as a medium for bonding to a polyoxyalkylene derivative having an oxyamino group.
In the methods for producing a polyoxyalkylene derivative having an oxyamino group which are described in JP-A-7-196925, International Publication No. 96/041813, and JP-T-2007-501811, in a first stage, it is necessary to convert the terminal hydroxyl group of the polyoxyalkylene obtained by polymerization of oxyalkylene to a functional group such as an activated ester or an amine and so on. In a second stage, the functionalized polyoxyalkylene is reacted and thereby attached with a low-molecular compound having a protected oxyamino group. In a third stage, the protected oxyamino group is deprotected to thereby obtain the desired product. The conventional production methods thus necessitate steps consisting of at least three stages, and purification by column chromatography, gel filtration, dialysis, etc. is performed in each stage, resulting in a considerably reduced yield and a prolonged production time. Because of this, those conventional methods are unsuitable for industrial production.
The polyoxyalkylene derivative described in U.S. Pat. No. 6,825,331 is obtained by a production method including introducing oxyphthalimide into the terminal hydroxyl group of a polyoxyalkylene using the Mitsunobu reaction, purifying the product by column chromatography, and then performing deprotection using a large amount of hydrazine. This production method results in a reduced yield due to purification by column chromatography, as stated above, and necessitates the use of a large amount of hydrazine, which is an inorganic base having poor handleability. It is therefore difficult to use this method for industrial production. It has also been ascertained that use of a polyoxyalkylene having a high molecular weight results in a decrease in purity.
As stated above, when the conventional production methods are used for producing a polyoxyalkylene having an oxyamino group, the number of production steps is large and there have been problems concerning, for example, a decrease in yield due to purification by column chromatography, etc. Furthermore, a high-molecular polyoxyalkylene having an oxyamino group is difficult to obtain as a high-purity product, and a product suitable for use as a modifying agent for bio-related substances has not been efficiently obtained so far.
As described above, there is a desire for a high-purity polyoxyalkylene derivative which has an oxyamino group and a high molecular weight and which can be effectively used in the modification of bio-related substances and can be industrially produced with ease.